Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

Irina Sadovskaya,Efstathios Giaouris,Mickael Meyrand,Douwe Van Sinderen,Pascal Courtin,Evgeny Vinogradov,Marie-Pierre Chapot-Chartier,Sylviane Furlan,Christine Péchoux,Saulius Kulakauskas,Jennifer Mahony,Julija Armalyte,Stuart Ainsworth,Simon Palussière,Yann Guérardel

doi:10.1128/mbio.01303-17

Abstract

ABSTRACTPolysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan.

Highlights

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall
In our previous study [11], cell wall-associated carbohydrates were extracted from the defatted cell walls of L. lactis MG1363 by trichloroacetic acid (TCA) treatment
We examined the Cell wall polysaccharides (CWPS) content of another strain, L. lactis 3107, that possesses a C-type CWPS biosynthesis cluster like MG1363 but belongs to the C2 subtype, whereas MG1363 belongs to the C1 subtype (Fig. 1)

Summary

Introduction

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In Gram-positive bacteria, members of the LytR-Cps2A-Psr protein family have been identified as the transferases involved in the anchoring of secondary cell wall glycopolymers, including WTA and CWPS (or capsular polysaccharide [CPS]) onto peptidoglycan [7,8,9,10]. We have previously reported that L. lactis MG1363 possesses a CWPS that constitutes a bacterial surface layer that was named the polysaccharide pellicle (PSP) [11, 12] This PSP is composed of branched hexasaccharide subunits that contain Glc, Gal, GlcNAc, and Rha and are linked by phosphodiester bonds. The PSP provides a protective barrier against phagocytosis by murine macrophages and acts as the receptor for members of various lactococcal phage groups, allowing their adsorption through specific recognition events [13,14,15]

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Conclusion